histone-DNA Interactions Research Articles

ChemInform Abstract: Mechanism(s) of SWI/SNF‐Induced Nucleosome Mobilization

AbstractReview: 92 refs.

The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4–DNA complexes

The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be the first and the last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) is a chaperone of the H3/H4 dimer that functions in both of these processes. However, little is known about the thermodynamics of chaperone–histone interactions or the direct role of Asf1 in the formation or disassembly of histone–DNA complexes. Here, we show that Saccharomyces cerevisiae Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone–DNA interactions through the formation of disomes. However, Asf1 was unable to disengage histones from DNA for tetrasomes formed with H3/H4 and strong nucleosome positioning DNA sequences or tetrasomes weakened by mutant (H3K56Q/H4) histones or non-positioning DNA sequences. Furthermore, Asf1 did not associate with preformed tetrasomes. These results are consistent with the measured affinity of Asf1 for H3/H4 dimers of 2.5 nM, which is weaker than the association of H3/H4 for DNA. These studies support a mechanism by which Asf1 aids H3/H4 deposition onto DNA but suggest that additional factors or post-translational modifications are required for Asf1 to remove H3/H4 from tetrasome intermediates in chromatin.

Unzipping Single DNA Molecules to Study the Structure of the Yeast Centromeric Nucleosome

Each centromere provides the essential locus for microtubule attachment on each chromosome during mitosis and meiosis in all eukaryotes. The centromeric nucleosome, the fundamental unit that makes up all centromeres, is distinct from conventional nucleosomes due to its lack of canonical H3 histones in place of a centromeric H3 histone variant (CenH3). Histone variants potentially change the structural properties of chromatin significantly and, therefore, impact chromosomal processes such as chromatid separation, pairing, etc. The structure of centromeric nucleosomes is widely debated, with main competing models proposed. In an effort to resolve the controversy, we have applied a unique single molecule technique that employs an optical trap to probe the force applied to unzip a single DNA molecule as its double strand is converted into two single strands. This produces a sequential map of the location of individual histone-DNA interactions with near base pair precision and accuracy. We have analyzed and compared the unzipping signatures of assembled recombinant canonical and centromeric yeast (Cse4) histones. Our results reveal features that are unique to centromeric structures in vitro and have implications for functional behavior in vivo.

The RSC Chromatin Remodeling ATPase can Translocate DNA with High Force and Small Step Size

ATP-dependent chromatin remodeling complexes (remodelers) use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodelers share highly conserved catalytic ATPase domains, many of which are shown to translocate DNA. Understanding remodeling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here we characterize the real-time activity of a minimal translocase ‘motor’ complex isolated from a prototypical remodeler (RSC) on bare DNA, using high-resolution optical tweezers and a ‘tethered’ translocase system. We observe on dsDNA a processivity of ∼35 bp, a speed of ∼25 bp/sec, and a step size of 1.9 (± 0.3, s.d.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA ‘buckling’ at initiation. These observations extend and clarify measurements of nucleosome-dependent translocation by the complete RSC or SWI/SNF complex, and reveal the ATPase as a powerful and versatile DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force using a small step size.

Deubiquitylase, DeSUMOylase, and DeISGylase Activity Microarrays for Assay of Substrate Preference and Functional Modifiers

Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.

Beads-on-String Structure of the Electrostatic Complex of DNA with a High-Generation PAMAM Dendrimer

The electrostatic complexes of polyanionic DNA with cationic dendrimer have been considered as a potential non-viral vector for gene delivery and a model system for understanding DNA-histone interaction. Although it is believed that the gene transfection efficiency may be influenced by the structure of the complex, the supramolecular structure of DNA-dendirmer complexes and its dependence on various system parameters such as dendrimer generation number, charge density, charge ratio and ionic strength are not well resolved. In this study, we investigate the structure of the complex of DNA with polyamidoamine (PAMAM) dendrimer of generation nine (G9) by means of synchrotron small angle X-ray scattering (SAXS). It is found that DNA is always able to wrap around the dendrimer to yield the beads-on-string structure irrespective of the charge density of the dendrimer. The effect of charge density on the persistence length of the chromatin-like fiber thus formed and the pitch length of the DNA superheix wrapping around the dendrimer are elucidated from the calculation of the SAXS profiles based on beads-on-string structure models. The first section in your paper

High-throughput sequencing reveals a simple model of nucleosome energetics

We use genome-wide nucleosome maps to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a minor role. Furthermore, similar sequence signatures are exhibited by control experiments in which nucleosome-free genomic DNA is either sonicated or digested with micrococcal nuclease, making it possible that current predictions based on high-throughput nucleosome-positioning maps are biased by experimental artifacts.

Mechanism(s) of SWI/SNF‐Induced Nucleosome Mobilization

Impediments to DNA access due to assembly of the eukaryotic genome into chromatin are in part overcome by the activity of ATP-dependent chromatin-remodeling complexes. These complexes employ energy derived from ATP hydrolysis to destabilize histone-DNA interactions and alter nucleosome positions, thereby increasing the accessibility of DNA-binding factors to their targets. However, the mechanism by which theses complexes accomplish this task remains unresolved. We review aspects of nucleosome alteration by the SWI/SNF complex, the archetypal remodeling enzyme. We focus on experiments that provide insights into how SWI/SNF induces nucleosome movement along DNA. Numerous biochemical activities have been characterized for this complex, all likely providing clues as to the molecular mechanism of translocation.

Nucleosome depletion at yeast terminators is not intrinsic and can occur by a transcriptional mechanism linked to 3'-end formation.

Genome-wide mapping of nucleosomes generated by micrococcal nuclease (MNase) suggests that yeast promoter and terminator regions are very depleted of nucleosomes, predominantly because their DNA sequences intrinsically disfavor nucleosome formation. However, MNase has strong DNA sequence specificity that favors cleavage at promoters and terminators and accounts for some of the correlation between occupancy patterns of nucleosomes assembled in vivo and in vitro. Using an improved method for measuring nucleosome occupancy in vivo that does not involve MNase, we confirm that promoter regions are strongly depleted of nucleosomes, but find that terminator regions are much less depleted than expected. Unlike at promoter regions, nucleosome occupancy at terminators is strongly correlated with the orientation of and distance to adjacent genes. In addition, nucleosome occupancy at terminators is strongly affected by growth conditions, indicating that it is not primarily determined by intrinsic histone-DNA interactions. Rapid removal of RNA polymerase II (pol II) causes increased nucleosome occupancy at terminators, strongly suggesting a transcription-based mechanism of nucleosome depletion. However, the distinct behavior of terminator regions and their corresponding coding regions suggests that nucleosome depletion at terminators is not simply associated with passage of pol II, but rather involves a distinct mechanism linked to 3'-end formation.

Weakly Positioned Nucleosomes Enhance the Transcriptional Competency of Chromatin

BackgroundTranscription is affected by nucleosomal resistance against polymerase passage. In turn, nucleosomal resistance is determined by DNA sequence, histone chaperones and remodeling enzymes. The contributions of these factors are widely debated: one recent title claims “… DNA-encoded nucleosome organization…” while another title states that “histone-DNA interactions are not the major determinant of nucleosome positions.” These opposing conclusions were drawn from similar experiments analyzed by idealized methods. We attempt to resolve this controversy to reveal nucleosomal competency for transcription.Methodology/Principal FindingsTo this end, we analyzed 26 in vivo, nonlinked, and in vitro genome-wide nucleosome maps/replicates by new, rigorous methods. Individual H2A nucleosomes are reconstituted inaccurately by transcription, chaperones and remodeling enzymes. At gene centers, weakly positioned nucleosome arrays facilitate rapid histone eviction and remodeling, easing polymerase passage. Fuzzy positioning is not due to artefacts. At the regional level, transcriptional competency is strongly influenced by intrinsic histone-DNA affinities. This is confirmed by reproducing the high in vivo occupancy of translated regions and the low occupancy of intergenic regions in reconstitutions from purified DNA and histones. Regional level occupancy patterns are protected from invading histones by nucleosome excluding sequences and barrier nucleosomes at gene boundaries and within genes.Conclusions/SignificanceDense arrays of weakly positioned nucleosomes appear to be necessary for transcription. Weak positioning at exons facilitates temporary remodeling, polymerase passage and hence the competency for transcription. At regional levels, the DNA sequence plays a major role in determining these features but positions of individual nucleosomes are typically modified by transcription, chaperones and enzymes. This competency is reduced at intergenic regions by sequence features, barrier nucleosomes, and proteins, preventing accessibility regulation of untargeted genes. This combination of DNA- and protein-influenced positioning regulates DNA accessibility and competence for regulatory protein binding and transcription. Interactive nucleosome displays are offered at http://chromatin.unl.edu/cgi-bin/skyline.cgi.

Nucleosome dynamics and epigenetic stability

Nucleosome remodelling is an essential principle to assure that the packaging of eukaryotic genomes in chromatin remains flexible and adaptable to regulatory needs. Nucleosome remodelling enzymes spend the energy of ATP to alter histone-DNA interactions, to catalyse nucleosome displacement and reassembly, on histone exchange and on the relocation of histone octamers on DNA. Despite these dynamics, chromatin structures encode 'epigenetic' information that governs the expression of the underlying genes. These information-bearing structures must be maintained over extended periods of time in resting cells and may be sufficiently stable to resist the turmoil of the cell cycle to be passed on to the next cell generation. Intuitively, nucleosome remodelling should antagonize the maintenance of stable structures. However, upon closer inspection it becomes evident that nucleosome remodelling is intimately involved in the assembly of stable chromatin structures that correspond to functional states. Remodellers may even contribute structural information themselves. Their involvement can be seen at several structural levels: at the levels of positioning individual nucleosomes, homoeostasis of linker histones, histone variants and non-histone proteins, as well as the differential folding of the nucleosome fibre. All of them may contribute to the assembly of heritable epigenetic structures.

Epigenetic control of plant immunity

In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone-DNA interactions. The genomic 'marks' that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid- and ethylene-sensitive defence mechanisms. ATP-dependent chromatin remodellers mediate the constitutive repression of the salicylic acid-dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial-infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions.

The DNA Sequence-dependence of Nucleosome Positioning in vivo and in vitro

The contribution of histone-DNA interactions to nucleosome positioning in vivo is currently a matter of debate. We argue here that certain nucleosome positions, often in promoter regions, in yeast may be, at least in part, specified by the DNA sequence. In contrast other positions may be poorly specified. Positioning thus has both statistical and DNA-determined components. We further argue that the relative affinity of the octamer for different DNA sequences can vary and therefore the interaction of histones with the DNA is a ‘tunable’ property.

Synergistic action of RNA polymerases in overcoming the nucleosomal barrier

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet must access the nucleosomal DNA. In vivo evidence suggests that multiple RNAPs might increase transcription efficiency through nucleosomes. Here we have quantitatively investigated this hypothesis using E. coli RNAP as a model system by directly monitoring its location on the DNA via a single molecule DNA unzipping technique. When an RNAP encountered a nucleosome, it paused with a distinctive 10-bp periodicity and was backtracked by ~10–15 bp. When two RNAPs elongated in close proximity, the trailing RNAP assisted the leading RNAP elongation, reducing its backtracking and enhancing its transcription through a nucleosome by a factor of 5. Taken together, our data indicate that histone-DNA interactions dictate RNAP pausing behavior, and alleviation of nucleosome-induced backtracking by multiple polymerases may serve as a mechanism for overcoming the nucleosomal barrier in vivo.

Mechanism of Chromatin Remodeling and Recovery During Passage of RNA Polymerase II

Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3/H4. The mechanism of survival and recovery of chromatin structure remains obscure. In our recent studies we have shown how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival. Structural modeling and functional analysis of the intermediates of transcription through a nucleosome was conducted. When Pol II approaches the area of strong DNA‐histone interactions, a small intranucleosomal DNA loop (zero‐size or Ø‐loop) containing transcribing enzyme is formed. During formation of the Ø‐loop, the recovery of DNA‐histone interactions behind Pol II is tightly coupled with their disruption ahead of the enzyme. This coupling is a distinct feature of the Pol II‐type mechanism that allows further transcription through the nucleosome, prevents nucleosome translocation and minimizes displacement of H3/H4 histones from DNA during enzyme passage.

The Histone Chaperone Nap1 Promotes Nucleosome Assembly by Eliminating Nonnucleosomal Histone DNA Interactions

The organization of the eukaryotic genome into nucleosomes dramatically affects the regulation of gene expression. The delicate balance between transcription and DNA compaction relies heavily on nucleosome dynamics. Surprisingly, little is known about the free energy required to assemble these large macromolecular complexes and maintain them under physiological conditions. Here, we describe the thermodynamic parameters that drive nucleosome formation in vitro. To demonstrate the versatility of our approach, we test the effect of DNA sequence and H3K56 acetylation on nucleosome thermodynamics. Furthermore, our studies reveal the mechanism of action of the histone chaperone nucleosome assembly protein 1 (Nap1). We present evidence for a paradigm in which nucleosome assembly requires the elimination of competing, nonnucleosomal histone-DNA interactions by Nap1. This observation is confirmed in vivo, wherein deletion of the NAP1 gene in yeast results in a significant increase in atypical histone-DNA complexes, as well as in deregulated transcription activation and repression.

Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer

Chromatin remodelers are sophisticated nano-machines that are able to alter histone-DNA interactions and to mobilize nucleosomes. Neither the mechanism of their action nor the conformation of the remodeled nucleosomes are, however, yet well understood. We have studied the mechanism of Remodels Structure of Chromatin (RSC)-nucleosome mobilization by using high-resolution microscopy and biochemical techniques. Atomic force microscopy and electron cryomicroscopy (EC-M) analyses show that two types of products are generated during the RSC remodeling: (i) stable non-mobilized particles, termed remosomes that contain about 180 bp of DNA associated with the histone octamer and, (ii) mobilized particles located at the end of DNA. EC-M reveals that individual remosomes exhibit a distinct, variable, highly-irregular DNA trajectory. The use of the unique "one pot assays" for studying the accessibility of nucleosomal DNA towards restriction enzymes, DNase I footprinting and ExoIII mapping demonstrate that the histone-DNA interactions within the remosomes are strongly perturbed, particularly in the vicinity of the nucleosome dyad. The data suggest a two-step mechanism of RSC-nucleosome remodeling consisting of an initial formation of a remosome followed by mobilization. In agreement with this model, we show experimentally that the remosomes are intermediate products generated during the first step of the remodeling reaction that are further efficiently mobilized by RSC.

Synergistic Action of RNA Polymerases in Overcoming the Nucleosomal Barrier

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet it must access the nucleosomal DNA. Various lines of in vivo evidence suggest that multiple RNAPs might increase transcription efficiency through nucleosomal DNA. Here we have quantitatively investigated this hypothesis by using E. coli RNAP as a model system and directly monitoring its location on the DNA via a single molecule DNA unzipping technique. When a single RNAP encountered a nucleosome, it paused with a distinctive 10-bp periodicity and was backtracked by an average distance of ∼10-15 bp. When two RNAPs were elongating in close proximity, the trailing RNAP exerted an assisting force on the leading RNAP, reducing its backtracking and enhancing its transcription through a nucleosome ∼5-fold. Taken together, our data indicate that histone-DNA interactions within a nucleosome dictate RNAP pausing behavior, and that alleviation of nucleosome-induced backtracking by multiple polymerases is a likely mechanism for overcoming the nucleosomal barrier in vivo.

RSC is an Efficient Nucleosome Randomizer: An AFM Quantitative Study on Oligo-Nucleosomal Templates

The fundamental unit of chromatin, the nucleosome, constitutes a barrier for several processes including transcription, repair and replication. One of the main tools that the cell uses to overcome this barrier is the recruitment of chromatin remodeling factors. The yeast RSC remodeling complex, a sophisticated nanomachine belonging to the SWI/SNF family, is able both to alter the histone-DNA interactions and to relocate nucleosomes. How RSC or the other complexes from the SWI/SNF family act on polynucleosomal templates is not fully understood and few quantitative data are available. Nevertheless, such quantification is required for both the understanding of the mechanism of action of RSC and the key role that it plays in the numerous vital processes for the cell.In order to gap this lack of quantitative information, we have used Atomic Force Microscopy (AFM) to visualize directly and at the single molecule level, the result of RSC action on oligo-nucleosomes. In parallel, we developed numerical simulations of the RSC sliding action, which quantitatively reproduce our experimental data. We demonstrate that RSC acts as an isotropic, processive and sequence-independent nucleosome randomizer. This multidisciplinary approach is very suitable to extend to other remodelers exhibiting different modes of action.

The Chromatin-Remodeling Complex ACF Functions as a Dimeric Motor to Space Nucleosomes

The chromatin structure at a given locus is a key determinant of its transcriptional state. Evenly spaced nucleosomes directly correlate with condensed chromatin structures and gene silencing. The ATP-dependent chromatin assembly factor (ACF) generates such structures in vitro and is required for transcriptional silencing in vivo. ACF generates and dynamically maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish such bidirectional movement is not known. Using FRET to follow disruption of histone-DNA interactions in real time we show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Employing Electron Paramagnetic Resonance (EPR) to resolve different populations of the nucleosome-ATPase complex, we find that the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Furthermore three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases bind facing each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome. Such a mechanism would allow rapid sampling of both sides of the nucleosome and allow bidirectional movement without dissociation. This novel dimeric motor mechanism differs from that of other dimeric motors such as kinesin and dimeric helicases that processively translocate in one direction and reflects the unique challenges faced by motors that move nucleosomes.